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<h1><span class="header-section-number"> 9</span> | Functional Gene Annotation with METABOLIC</h1>
<p><font size="1"><strong>Created by:</strong> Caitlin Casar on 2021-01-20 </br>
<strong>Last updated:</strong> 2021-01-20 </font></p>
<p>This is a protocol for annotating your metagenome or binned genome data with <a href="https://github.com/AnantharamanLab/METABOLIC" target="_blank">METABOLIC</a>.</p>
<p>First you will need to install METABOLIC on Quest. Save the following to a .sh file to your home directory and run as a batch job on Quest:</p>
<div class="sourceCode" id="cb30"><pre class="sourceCode bash"><code class="sourceCode bash"><a class="sourceLine" id="cb30-1" title="1"><span class="ex">module</span> load anaconda3</a>
<a class="sourceLine" id="cb30-2" title="2"></a>
<a class="sourceLine" id="cb30-3" title="3"><span class="co"># create env</span></a>
<a class="sourceLine" id="cb30-4" title="4"><span class="ex">conda</span> create -n metabolic</a>
<a class="sourceLine" id="cb30-5" title="5"><span class="bu">source</span> activate metabolic</a>
<a class="sourceLine" id="cb30-6" title="6"></a>
<a class="sourceLine" id="cb30-7" title="7"><span class="co"># conda install the required tools</span></a>
<a class="sourceLine" id="cb30-8" title="8"><span class="ex">conda</span> install sambamba</a>
<a class="sourceLine" id="cb30-9" title="9"><span class="ex">conda</span> install bamtools</a>
<a class="sourceLine" id="cb30-10" title="10"><span class="ex">conda</span> install coverm <span class="co"># installs perl 5.32</span></a>
<a class="sourceLine" id="cb30-11" title="11"><span class="ex">conda</span> install gtdbtk</a>
<a class="sourceLine" id="cb30-12" title="12"><span class="ex">conda</span> install diamond</a>
<a class="sourceLine" id="cb30-13" title="13"><span class="ex">conda</span> install bowtie2</a>
<a class="sourceLine" id="cb30-14" title="14"><span class="ex">conda</span> install R=3.6.0</a>
<a class="sourceLine" id="cb30-15" title="15"></a>
<a class="sourceLine" id="cb30-16" title="16"><span class="co"># conda install R dependencies</span></a>
<a class="sourceLine" id="cb30-17" title="17"><span class="ex">conda</span> install r-tidyverse=1.3.0</a>
<a class="sourceLine" id="cb30-18" title="18"><span class="ex">conda</span> install r-diagram</a>
<a class="sourceLine" id="cb30-19" title="19"><span class="ex">conda</span> install r-ggthemes</a>
<a class="sourceLine" id="cb30-20" title="20"><span class="ex">conda</span> install r-ggalluvial</a>
<a class="sourceLine" id="cb30-21" title="21"><span class="ex">conda</span> install r-ggraph</a>
<a class="sourceLine" id="cb30-22" title="22"><span class="ex">conda</span> install r-openxlsx</a>
<a class="sourceLine" id="cb30-23" title="23"><span class="ex">conda</span> install r-pdftools</a>
<a class="sourceLine" id="cb30-24" title="24"></a>
<a class="sourceLine" id="cb30-25" title="25"><span class="co"># conda install perl dependencies</span></a>
<a class="sourceLine" id="cb30-26" title="26"><span class="ex">conda</span> install perl-data-dumper <span class="co"># downgrades perl to 5.26.2</span></a>
<a class="sourceLine" id="cb30-27" title="27"><span class="ex">conda</span> install perl-excel-writer-xlsx</a>
<a class="sourceLine" id="cb30-28" title="28"><span class="ex">conda</span> install perl-posix</a>
<a class="sourceLine" id="cb30-29" title="29"><span class="ex">conda</span> install perl-getopt-long</a>
<a class="sourceLine" id="cb30-30" title="30"><span class="ex">conda</span> install perl-statistics-descriptive</a>
<a class="sourceLine" id="cb30-31" title="31"><span class="ex">conda</span> install perl-bioperl</a>
<a class="sourceLine" id="cb30-32" title="32"></a>
<a class="sourceLine" id="cb30-33" title="33"><span class="co"># get the one pesky perl dependency not available through conda</span></a>
<a class="sourceLine" id="cb30-34" title="34"><span class="ex">conda</span> install perl-app-cpanminus</a>
<a class="sourceLine" id="cb30-35" title="35"><span class="fu">env</span> PERL5LIB=<span class="st">""</span> PERL_LOCAL_LIB_ROOT=<span class="st">""</span> PERL_MM_OPT=<span class="st">""</span> PERL_MB_OPT=<span class="st">""</span> cpanm Array::Split</a>
<a class="sourceLine" id="cb30-36" title="36"></a>
<a class="sourceLine" id="cb30-37" title="37"><span class="co">#conda install gdown</span></a>
<a class="sourceLine" id="cb30-38" title="38"><span class="ex">conda</span> install gdown</a>
<a class="sourceLine" id="cb30-39" title="39"></a>
<a class="sourceLine" id="cb30-40" title="40"><span class="co"># conda install the perl package to solve the first (and so far only) error</span></a>
<a class="sourceLine" id="cb30-41" title="41"><span class="ex">conda</span> install perl-parallel-forkmanager</a>
<a class="sourceLine" id="cb30-42" title="42"></a>
<a class="sourceLine" id="cb30-43" title="43"><span class="co">#clone github repo</span></a>
<a class="sourceLine" id="cb30-44" title="44"><span class="fu">git</span> clone https://github.com/AnantharamanLab/METABOLIC.git</a>
<a class="sourceLine" id="cb30-45" title="45"></a>
<a class="sourceLine" id="cb30-46" title="46"><span class="co">#run setup script - make sure you are working from your metabolic conda environment or this script will fail</span></a>
<a class="sourceLine" id="cb30-47" title="47"><span class="bu">cd</span> METABOLIC</a>
<a class="sourceLine" id="cb30-48" title="48"><span class="fu">sh</span> run_to_setup.sh</a>
<a class="sourceLine" id="cb30-49" title="49"></a>
<a class="sourceLine" id="cb30-50" title="50"><span class="co">#test installation - this is optional but good for troubleshooting installation errors</span></a>
<a class="sourceLine" id="cb30-51" title="51"><span class="fu">perl</span> /home/cpc7770/METABOLIC/METABOLIC-G.pl -test true</a>
<a class="sourceLine" id="cb30-52" title="52"><span class="fu">perl</span> /home/cpc7770/METABOLIC/METABOLIC-C.pl -test true</a></code></pre></div>
<p>Now you’re ready to run METABOLIC on your metagenome data. If you want to run it on unbinned metagenome data, follow this example batch script. Note that for <code>METABOLIC-G.pl</code> you only need to specify the input and output directory. You can run it on either nucleotide files with a <code>.fasta</code> extension, or amino acid files with a <code>.faa</code> extension.</p>
<div class="sourceCode" id="cb31"><pre class="sourceCode bash"><code class="sourceCode bash"><a class="sourceLine" id="cb31-1" title="1"><span class="ex">module</span> load anaconda3</a>
<a class="sourceLine" id="cb31-2" title="2"><span class="bu">source</span> activate metabolic</a>
<a class="sourceLine" id="cb31-3" title="3"></a>
<a class="sourceLine" id="cb31-4" title="4"><span class="co">#run on metagenomes - note that the file extensions MUST be '.fasta', '.fa' is not acceptable.</span></a>
<a class="sourceLine" id="cb31-5" title="5"><span class="fu">perl</span> /home/cpc7770/METABOLIC/METABOLIC-G.pl -in-gn /projects/p30777/metagenome_data/contigs -o /projects/p30777/metagenome_data/metabolic_metagenome_annotations</a></code></pre></div>
<p>Wanna run it on binned genomes? Use the <code>METABOLIC-C.pl</code> script. Note that for this script you need to specify the paired read files. You can run the following as a batch job on Quest:</p>
<div class="sourceCode" id="cb32"><pre class="sourceCode bash"><code class="sourceCode bash"><a class="sourceLine" id="cb32-1" title="1"><span class="co">#run on binned genomes</span></a>
<a class="sourceLine" id="cb32-2" title="2"></a>
<a class="sourceLine" id="cb32-3" title="3"><span class="co">#rename from .fa to .fasta</span></a>
<a class="sourceLine" id="cb32-4" title="4"><span class="co">#cd /projects/p30777/metagenome_data/genomes</span></a>
<a class="sourceLine" id="cb32-5" title="5"><span class="co">#find . -name "*.fa" -exec rename .fa .fasta {} +</span></a>
<a class="sourceLine" id="cb32-6" title="6"></a>
<a class="sourceLine" id="cb32-7" title="7"></a>
<a class="sourceLine" id="cb32-8" title="8"><span class="co">#run on metagenomes - note that the file extensions MUST be '.fasta', '.fa' is not acceptable.</span></a>
<a class="sourceLine" id="cb32-9" title="9"><span class="co">#loop over directories </span></a>
<a class="sourceLine" id="cb32-10" title="10"><span class="kw">for</span> <span class="ex">path</span> in /projects/p30777/metagenome_data/genomes/*<span class="kw">;</span> <span class="kw">do</span></a>
<a class="sourceLine" id="cb32-11" title="11"> [ <span class="ex">-d</span> <span class="st">"</span><span class="va">${path}</span><span class="st">"</span> ] <span class="kw">||</span> <span class="bu">continue</span> <span class="co"># if not a directory, skip</span></a>
<a class="sourceLine" id="cb32-12" title="12"> <span class="va">dirname=</span><span class="st">"</span><span class="va">$(</span><span class="fu">basename</span> <span class="st">"</span><span class="va">${path}</span><span class="st">"</span><span class="va">)</span><span class="st">"</span></a>
<a class="sourceLine" id="cb32-13" title="13"> <span class="bu">echo</span> annotating <span class="va">$dirname</span> genomes...</a>
<a class="sourceLine" id="cb32-14" title="14"> <span class="fu">perl</span> ~/METABOLIC/METABOLIC-C.pl -in-gn <span class="va">$path</span> -r /projects/p30777/metagenome_data/reads/<span class="va">$dirname</span> -o /projects/p30777/metagenome_data/metabolic_genome_annotations/<span class="va">$dirname</span></a>
<a class="sourceLine" id="cb32-15" title="15"><span class="kw">done</span></a>
<a class="sourceLine" id="cb32-16" title="16"></a>
<a class="sourceLine" id="cb32-17" title="17"><span class="bu">echo</span> done!</a></code></pre></div>
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